Angiotensin-converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1, ACE) is a zinc-dependent dipeptidyl carboxypeptidase with diverse physiological functions, including that of blood pressure regulation via angiotensin II production and bradykinin inactivation.
Somatic ACE (sACE), a type I transmembrane protein, is composed of two homologous catalytic domains (N- (SEQ ID NO: 1) and C- domains) arising from a gene duplication event (Soubrier et al., 1988). The germinal form of ACE (testis ACE, (tACE) SEQ ID NO: 2) (Ehlers et al., 1989) originates from the same gene as sACE , but has a tissue-specific promoter located within intron 12. Testis ACE (SEQ ID NO: 2) plays a crucial role in reproduction (Hagaman et al., 1998).
Despite sharing ˜60% sequence identity with the C-domain, the N-domain has its own distinctive physicochemical and functional properties. It is thermally more stable than the C-domain (Voronov et al., 2002), more resistant to proteolysis under denaturing conditions and is less dependent on chloride activation relative to the C-domain (Wei et al., 1991; Jaspard et al., 1993). The N- and the C-domains are joined by a linker that is susceptible to proteolysis (Sturrock et al., (1997), Biochem. Biophys. Res. 236, 16-19). It has also been suggested that the N- and the C-domains have unique physiological roles and that they have negative effect on each other (Woodman et al., 2005).
Substrates such as the hemoregulatory peptide AcSDKP (Rousseau et al., 1995), angiotensin 1-7 (Deddish et al., 1998), and the enkephalin precursor Met5-Enk-Arg6-Phe7 (Deddish et al., 1997) are specific for the N-domain, whereas the physiological substrates bradykinin and angiotensin I are hydrolysed with similar catalytic efficiency as compared with the C-domain.
It has been reported that the N-domain preferentially hydrolyses the A beta peptide of the amyloid precursor protein resulting in inhibition of A beta aggregation and cytotoxicity (Oba et al., 2005). The widely-used ACE inhibitor captopril shows modest selectivity for the N-domain (Wei et al., 1992); however, the phosphinic peptide inhibitor RXP-407 has a dissociation constant three orders of magnitude lower for the N-domain of the enzyme (Dive et al., 1999).
The N-domain has 10 N-linked sites of which 7 are unique to the N-domain. The different glycan profile of the N-domain is likely responsible for the carbohydrate-mediated dimerisation of sACE which has been described under certain conditions (Kost et al., 2003). Moreover, the difference in glycosylation could impact on the structural basis for epitope recognition and epitope mapping of the N-domain has revealed a region that might play a role in the relatively inefficient ectodomain shedding of sACE compared to its germinal isoform (Balyasnikova et al., 2005).
Both the N- and the C-domains of ACE protein are heavily glycosylated in nature, a feature that has hampered 3D structural determination of the protein and of each of the domains.
We previously described the 3D structure of the ACE protein (International Patent Application PCT/GB03/03966 (published as WO 04/024765). This 3D structure was that of the underglycosylated C-domain of ACE protein.
This underglycosylated 3D structure, however, provides limited information on the structure of the N-domain of ACE, nor is it ideal for screening or designing domain specific modulators suitable for pharmaceutical use nor indeed for studying the functional interaction between the N- and the C-domains of ACE protein.
Therefore there is a need to obtain a crystal of the N-domain of ACE protein with sufficient quality to allow crystallographic data to be obtained. Further, there is a need for such a crystal to allow the determination of the crystal structure of the N-domain of ACE. Finally there is a need for procedures for studying the interplay between the N- and the C-domains and screening for domain specific modulators using the 3D structural information of the N-domain of ACE protein.